US20100204277A1 - Compositions and Methods for Controlling Infestation - Google Patents

Compositions and Methods for Controlling Infestation Download PDF

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Publication number
US20100204277A1
US20100204277A1 US12/594,688 US59468807A US2010204277A1 US 20100204277 A1 US20100204277 A1 US 20100204277A1 US 59468807 A US59468807 A US 59468807A US 2010204277 A1 US2010204277 A1 US 2010204277A1
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alkyl
hydrogen
independently selected
branched
eggs
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Vernon Morrison Bowles
Paul MacLeman
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Dr Reddys Laboratories SA
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Hatchtech Pty Ltd
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
    • A01N43/34—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one nitrogen atom as the only ring hetero atom
    • A01N43/40—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one nitrogen atom as the only ring hetero atom six-membered rings
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N35/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having two bonds to hetero atoms with at the most one bond to halogen, e.g. aldehyde radical
    • A01N35/04—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having two bonds to hetero atoms with at the most one bond to halogen, e.g. aldehyde radical containing aldehyde or keto groups, or thio analogues thereof, directly attached to an aromatic ring system, e.g. acetophenone; Derivatives thereof, e.g. acetals
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N35/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having two bonds to hetero atoms with at the most one bond to halogen, e.g. aldehyde radical
    • A01N35/08—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having two bonds to hetero atoms with at the most one bond to halogen, e.g. aldehyde radical at least one of the bonds to hetero atoms is to nitrogen
    • A01N35/10—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having two bonds to hetero atoms with at the most one bond to halogen, e.g. aldehyde radical at least one of the bonds to hetero atoms is to nitrogen containing a carbon-to-nitrogen double bond
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
    • A01N43/34—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one nitrogen atom as the only ring hetero atom
    • A01N43/40—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one nitrogen atom as the only ring hetero atom six-membered rings
    • A01N43/42—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one nitrogen atom as the only ring hetero atom six-membered rings condensed with carbocyclic rings
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
    • A01N43/90—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having two or more relevant hetero rings, condensed among themselves or with a common carbocyclic ring system
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
    • Y10T436/00—Chemistry: analytical and immunological testing
    • Y10T436/14—Heterocyclic carbon compound [i.e., O, S, N, Se, Te, as only ring hetero atom]
    • Y10T436/145555—Hetero-N

Definitions

  • the present invention relates to compositions and methods for inhibiting the activity of an enzyme or enzymes that are directly or indirectly involved in invertebrate remodelling events.
  • the invention relates to compositions and methods for controlling invertebrate multicellular organisms having cross-linked protein structures that include but are not limited to eggs, sheaths, carapaces, exoskeletons, cysts, cocoons or ootheca.
  • the invention also provides methods of inhibiting processes such as apolysis, ecdysis, egg hatching, excystment, exsheathment and metamorphosis.
  • the invention also provides methods and compositions for preventing, treating or controlling infestations of an invertebrate pest that undergoes remodelling events.
  • Pests that undergo remodelling events such as egg hatching, moulting and/or metamorphosis from pupae to adult, cause significant problems in a wide variety of situations.
  • pests that undergo such remodelling events may externally infest humans or animals and annoy, bite and/or cause infections, particularly of humans and domesticated animals. These pests may also internally infest humans and animals causing infection, gastrointestinal problems, swelling, and/or lymphatic problems and blood loss.
  • Pests that undergo remodelling events may also infest plants and their larvae or other life cycle stages can eat leaves, flowers, roots and fruit causing significant damage to commercially important crops.
  • Other pests that undergo these remodelling events infest the environment and cause illness to humans or animals or property damage. For example, termites cause significant property damage and the presence of dust or house mites can cause asthma in humans.
  • pesticides are known for controlling or eliminating plant, human, animal and environmental pests. These pesticides may be used in the form of aerosols, space sprays, liquids, soaps, shampoos, wettable powders, granules, baits, dusts, tablets and the like.
  • Conventional control methods for pests rely on the use of chemical pesticides such as chlorinated hydrocarbons (DDT, endosulfan, etc.), synthetic and natural pyrethrins (pyrethrin, permethrin, cypermethrin, deltamethrin), insect growth regulators that are known to interfere with chitin synthesis, insecticidal bacterial toxins ( Bacillus thuringiensis (Bt) toxins) and nematicides including both fumigant and non-fumigant (ie formulated granules or liquids).
  • chemical pesticides such as chlorinated hydrocarbons (DDT, endosulfan, etc.), synthetic and natural pyrethrins (pyrethrin, permethrin, cypermethrin, deltamethrin), insect growth regulators that are known to interfere with chitin synthesis, insecticidal bacterial toxins ( Bacillus thuringiensis (Bt) toxins) and
  • the present invention is directed to methods of treating pest infestation by inhibiting metabolic processes of the pest such as for example, processes involved in invertebrate remodelling.
  • the methods of the invention comprise decreasing exsheathment of an invertebrate by externally contacting a pest with a compound of formula (I):
  • X is selected from a covalent bond, —C(R 5 ) 2 —, —Z— or —C(R 5 ) 2 —Z—C(R 5 ) 2 —;
  • R 1 and R 1′ are independently selected from hydrogen, C 1-6 alkyl, a branched-chain C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, hydroxy, C 1-6 alkoxy, thiol, C 1-6 alkylthio, halogen, C(R 6 ) 3 , CO 2 H, CO 2 C 1-6 alkyl, SO 3 H, SO 3 C 1-6 alkyl, NH 2 , NHC 1-6 alkyl or N(C 1-6 alkyl) 2 , or R 1 and R 19 taken together are —C(R 5 ) 2 —, —C(R 5 ) 2 —C(R 5 ) 2 —, —CR 5 ⁇ CR 5 —, C(O), C(S) or NH;
  • R 2 , R 2′ , R 3 , R 3′ , R 4 and R 4′ are independently selected from hydrogen, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, hydroxy, C 1-6 alkoxy, thiol, C 1-6 alkylthiol, halogen, CN, C(R 6 ) 3 , CO 2 H, CO 2 C 1-6 alkyl, SO 3 H, SO 3 C 1-6 alkyl, NH 2 , NHC 1-6 alkyl or N(C 1-6 alkyl) 2 , —CH 2 CHNH(CO 2 H), NH(C 1-6 alkylene)N(C 1-6 alkyl) 2 or a 5 or 6 membered carbocyclic or heterocyclic ring; or
  • each R 5 is independently selected from hydrogen, C 1-6 alkyl, a branched-chain C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, hydroxy, C 1-6 alkoxy, thiol, C 1-6 alkylthiol, CO 2 H, CO 2 C 1-6 alkyl, SO 3 H, SO 3 C 1-6 alkyl, NH 2 , NHC 1-6 alkyl or N(C 1-6 alkyl) 2 ;
  • each R 6 is independently selected from hydrogen and halogen
  • Z is selected from a covalent bond, —NH—, —O—, —S—, —C(O)— and —C(S)—;
  • a pharmaceutically, veterinary or agriculturally acceptable salt thereof in an amount effective to inhibit or decrease the rate of exsheathment of said invertebrate.
  • X is selected from a covalent bond, —C(R 5 ) 2 —, —Z—or —C(R 5 ) 2 —Z—C(R 5 ) 2 —;
  • R 1 and R 1′ are independently selected from hydrogen, C 1-6 alkyl, a branched-chain C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, hydroxy, C 1-6 alkoxy, thiol, C 1-6 alkylthio, halogen, C(R 6 ) 3 , CO 2 H, CO 2 C 1-6 alkyl, SO 3 H, SO 3 C 1-6 alkyl, NH 2 , NHC 1-6 alkyl or N(C 1-6 alkyl) 2 , or R 1 and R 19 taken together are —C(R 5 ) 2 —, —C(R 5 ) 2 —C(R 5 ) 2 —, —CR 5 ⁇ CR 5 —, C(O), C(S) or NH;
  • R 2 , R 2′ , R 3 , R 3′ , R 4 and R 4′ are independently selected from hydrogen, C 1-6 alkyl, a branched-chain C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, hydroxy, C 1-6 alkoxy, thiol, C 1-6 alkylthiol, halogen, CN, C(R 6 ) 3 , CO 2 H, CO 2 C 1-6 alkyl, SO 3 H, SO 3 C 1-6 alkyl, NH 2 , NHC 1-9 alkyl or N(C 1-6 alkyl) 2 , —CH 2 CHNH(CO 2 H), NH(C 1-6 alkylene)N(C 1-6 alkyl) 2 or a 5 or 6 membered carbocyclic or heterocyclic ring; or
  • each R 5 is independently selected from hydrogen, C 1-6 alkyl, a branched-chain C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, hydroxy, C 1-6 alkoxy, thiol, C 1-6 alkylthiol, CO 2 H, CO 2 C 1-6 alkyl, SO 3 H, SO 3 C 1-6 alkyl, NH 2 , NHC 1-6 alkyl or N(C 1-6 alkyl) 2 ;
  • each R 6 is independently selected from hydrogen and halogen
  • Z is selected from a covalent bond, —NH—, —O—, —S—, —C(O)— and —C(S)—;
  • a pharmaceutically, veterinary or agriculturally acceptable salt thereof in an amount effective to inhibit or otherwise decrease the rate of excystment of said invertebrate.
  • the invention further contemplates methods of treating pest infestation comprising decreasing apolysis of an invertebrate by externally contacting a pest with a compound of formula:
  • X is selected from a covalent bond, —C(R 5 ) 2 —, —Z— or —C(R 5 ) 2 —Z—C(R 5 ) 2 —;
  • R 1 and R 1′ are independently selected from hydrogen, C 1-6 alkyl, a branched-chain C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, hydroxy, C 1-6 alkoxy, thiol, C 1-6 alkylthio, halogen, C(R 6 ) 3 , CO 2 H, CO 2 C 1-6 alkyl, SO 3 H, SO 3 C 1-6 alkyl, NH 2 , NHC 1-6 alkyl or N(C 1-6 alkyl) 2 , or R 1 and R 19 taken together are —C(R 5 ) 2 —, —C(R 5 ) 2 —C(R 5 ) 2 —, —CR 5 ⁇ CR 5 —, C(O), C(S) or NH;
  • R 2 , R 2′ , R 3 , R 3′ , R 4 and R 4′ are independently selected from hydrogen, C 1-6 alkyl, a branched-chain C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, hydroxy, C 1-6 alkoxy, thiol, C 1-6 alkylthiol, halogen, CN, C(R 6 ) 3 , CO 2 H, CO 2 C 1-6 alkyl, SO 3 H, SO 3 C 1-6 alkyl, NH 2 , NHC 1-6 alkyl or N(C 1-6 alkyl) 2 , —CH 2 CHNH(CO 2 H), NH(C 1-6 alkylene)N(C 1-6 alkyl) 2 or a 5 or 6 membered carbocyclic or heterocyclic ring; or
  • each R 5 is independently selected from hydrogen, C 1-6 alkyl, a branched-chain C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, hydroxy, C 1-6 alkoxy, thiol, C 1-6 alkylthiol, CO 2 H, CO 2 C 1-6 alkyl, SO 3 H, SO 3 C 1-6 alkyl, NH 2 , NHC 1-6 alkyl or N(C 1-6 alkyl) 2 ;
  • each R 6 is independently selected from hydrogen and halogen
  • Z is selected from a covalent bond, —NH—, —O—, —S—, —C(O)— and —C(S)—;
  • a pharmaceutically, veterinary or agriculturally acceptable salt thereof in an amount effective to inhibit or otherwise decrease the rate of apolysis of said invertebrate.
  • the methods of the invention involve treating pest infestation comprising inhibiting metamorphosis of an invertebrate by externally contacting said pest with a compound of formula:
  • X is selected from a covalent bond, —C(R 5 ) 2 —, —Z— or —C(R 5 ) 2 —Z—C(R 5 ) 2 —;
  • R 1 and R 1′ are independently selected from hydrogen, C 1-6 alkyl, a branched-chain C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, hydroxy, C 1-6 alkoxy, thiol, C 1-6 alkylthio, halogen, C(R 6 ) 3 , CO 2 H, CO 2 C 1-6 alkyl, SO 3 H, SO 3 C 1-6 alkyl, NH 2 , NHC 1-6 alkyl or N(C 1-6 alkyl) 2 , or R 1 and R 19 taken together are —C(R 5 ) 2 —, —C(R 5 ) 2 —C(R 5 ) 2 —, —CR 5 ⁇ CR 5 —, C(O), C(S) or NH;
  • R 2 , R 2′ , R 3 , R 3′ , R 4 and R 4′ are independently selected from hydrogen, C 1-6 alkyl, a branched-chain C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, hydroxy, C 1-6 alkoxy, thiol, C 1-6 alkylthiol, halogen, CN, C(R 6 ) 3 , CO 2 H, CO 2 C 1-6 alkyl, SO 3 H, SO 3 C 1-6 alkyl, NH 2 , NHC 1-6 alkyl or N(C 1-6 alkyl) 2 , —CH 2 CHNH(CO 2 H), NH(C 1-6 alkylene)N(C 1-6 alkyl) 2 or a 5 or 6 membered carbocyclic or heterocyclic ring; or
  • each R 5 is independently selected from hydrogen, C 1-6 alkyl, a branched-chain C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, hydroxy, C 1-6 alkoxy, thiol, C 1-6 alkylthiol, CO 2 H, CO 2 C 1-6 alkyl, SO 3 H, SO 3 C 1-6 alkyl, NH 2 , NHC 1-6 alkyl or N(C 1-6 alkyl) 2 ;
  • each R 6 is independently selected from hydrogen and halogen
  • Z is selected from a covalent bond, —NH—, —O—, —S—, —C(O)— and —C(S)—;
  • the compound is a metal chelating agent, wherein the metal chelating agent has at least two polar atoms capable of simultaneously coordinating with a metal ion, has a clogP value of /1 and ⁇ 4; and/or and a molar refractivity in the range of 40 to 90 cm 3 /mole.
  • the metal chelating agent is not 1,10-phenanthroline. In other embodiments, the metal chelating agent is not a dipyridyl compound.
  • the methods of the invention contemplate the use of multiple pesticides and interventions for treating infestations.
  • the methods further comprises contacting said pest with a second pesticide.
  • aspects of the invention comprise methods of treating pest infestation comprising decreasing exsheathment of an invertebrate by externally contacting a pest with a compound of formula (II):
  • R 27 is C 1-6 alkyl, a branched-chain C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, hydroxy, C 1-6 alkoxy, thiol, C 1-6 alkylthio, C(R 29 ) 2 , N(R 30 ) 2 , or a 5 or 6 membered carbocyclic ring or heterocyclic ring;
  • Still another embodiment of the invention describes a method of treating pest infestation comprising decreasing excystment of an invertebrate by externally contacting a pest with a compound of formula (II):
  • the invention also contemplates methods of treating pest infestation comprising decreasing apolysis of an invertebrate by externally contacting a pest with a compound of formula (II):
  • Yet another alternative method of treating pest infestation comprises inhibiting metamorphosis of an invertebrate by externally contacting said pest with a compound of formula:
  • the compound is preferably a metal chelating agent, wherein the metal chelating agent has at least two polar atoms capable of simultaneously coordinating with a metal ion, has a clogP value of /1 and ⁇ 4; and/or and a molar refractivity in the range of 40 to 90 cm 3 /mole.
  • the metal chelating agent is not 1,10-phenanthroline.
  • the foregoing methods may further comprise contacting the pest with a second, third, fourth or more pesticides. Further, the pest may be treated multiple times with the various pesticides described herein.
  • the methods described herein produce a greater decrease in the rate of exsheathment, excystment, apolysis or metamorphosis than is observed with the administration of 1,10 phenanthroline.
  • the methods are employed for killing an invertebrate pest, said method comprising externally contacting said pest with a compound of formula (I):
  • X is selected from a covalent bond, —C(R 5 ) 2 —, —Z— or —C(R 5 ) 2 —Z—C(R 5 ) 2 —;
  • R 1 and R 1′ are independently selected from hydrogen, C 1-6 alkyl, a branched-chain C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, hydroxy, C 1-6 alkoxy, thiol, C 1-6 alkylthio, halogen, C(R 6 ) 3 , CO 2 H, CO 2 C 1-6 alkyl, SO 3 H, SO 3 C 1-6 alkyl, NH 2 , NHC 1-6 alkyl or N(C 1-6 alkyl) 2 , or R 1 and R 19 taken together are —C(R 5 ) 2 —, —C(R 5 ) 2 —C(R 5 ) 2 —, —CR 5 ⁇ CR 5 —, C(O), C(S) or NH;
  • R 2 , R 2′ , R 3 , R 3′ , R 4 and R 4′ are independently selected from hydrogen, C 1-6 alkyl, a branched-chain C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, hydroxy, C 1-6 alkoxy, thiol, C 1-6 alkylthiol, halogen, CN, C(R 6 ) 3 , CO 2 H, CO 2 C 1-6 alkyl, SO 3 H, SO 3 C 1-6 alkyl, NH 2 , NHC 1-6 alkyl or N(C 1-6 alkyl) 2 , —CH 2 CHNH(CO 2 H), NH(C 1-6 alkylene)N(C 1-6 alkyl) 2 or a 5 or 6 membered carbocyclic or heterocyclic ring; or
  • each R 5 is independently selected from hydrogen, C 1-6 alkyl, a branched-chain C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, hydroxy, C 1-6 alkoxy, thiol, C 1-6 alkylthiol, CO 2 H, CO 2 C 1-6 alkyl, SO 3 H, SO 3 C 1-6 alkyl, NH 2 , NHC 1-6 alkyl or N(C 1-6 alkyl) 2 ;
  • each R 6 is independently selected from hydrogen and halogen
  • Z is selected from a covalent bond, —NH—, —O—, —S—, —C(O)— and —C(S)—;
  • inventions are directed to methods of killing an invertebrate pest, said method comprising externally contacting said pest with a compound of formula (II):
  • Y 1 and Y 2 are independently selected form O, NR 28 , or S;
  • the invertebrate pest is selected from the group consisting of nematodes, trematodes, cestodes, lice, fleas, mites and scabies, moths, beetles, caterpillars butterflies, termites, arachnids, cockroaches, centipedes, fleas and mites.
  • the methods are such that they are used to kill at least some of the invertebrate pests that are infesting a host. In preferred embodiments, the methods produce results in which at least 25% of the pests in a given infestation are killed. In other embodiments, at least 30%, of the pest population is killed. In still other embodiments, at least 50% of the invertebrate population in a given infestation is killed. In still other preferred embodiments, at least 75% of the invertebrate pest population in a given infestation is killed.
  • Also contemplated are methods of inhibiting a remodelling event in an invertebrate population comprising contacting said invertebrate population with a compound of formula (I):
  • X is selected from a covalent bond, —C(R 5 ) 2 —, —Z— or —C(R 5 ) 2 —Z—C(R 5 ) 2 —;
  • R 1 and R 1′ are independently selected from hydrogen, C 1-6 alkyl, a branched-chain C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, hydroxy, C 1-6 alkoxy, thiol, C 1-6 alkylthio, halogen, C(R 6 ) 3 , CO 2 H, CO 2 C 1-6 alkyl, SO 3 H, SO 3 C 1-6 alkyl, NH 2 , NHC 1-6 alkyl or N(C 1-6 alkyl) 2 , or R 1 and R 19 taken together are —C(R 5 ) 2 —, —C(R 5 ) 2 —C(R 5 ) 2 —, —CR 5 ⁇ CR 5 —, C(O), C(S) or NH;
  • R 2 , R 2′ , R 3 , R 3′ , R 4 and R 4′ are independently selected from hydrogen, C 1-6 alkyl, a branched-chain C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, hydroxy, C 1-6 alkoxy, thiol, C 1-6 alkylthiol, halogen, CN, C(R 6 ) 3 , CO 2 H, CO 2 C 1-6 alkyl, SO 3 H, SO 3 C 1-6 alkyl, NH 2 , NHC 1-6 alkyl or N(C 1-6 alkyl) 2 , —CH 2 CHNH(CO 2 H), NH(C 1-6 alkylene)N(C 1-6 alkyl) 2 or a 5 or 6 membered carbocyclic or heterocyclic ring; or
  • each R 5 is independently selected from hydrogen, C 1-6 alkyl, a branched-chain C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, hydroxy, C 1-6 alkoxy, thiol, C 1-6 alkylthiol, CO 2 H, CO 2 C 1-6 alkyl, SO 3 H, SO 3 C 1-6 alkyl, NH 2 , NHC 1-6 alkyl or N(C 1-6 alkyl) 2 ;
  • each R 6 is independently selected from hydrogen and halogen
  • Z is selected from a covalent bond, —NH—, —O—, —S—, —C(O)— and —C(S)—;
  • invertebrate remodelling event is not egg hatching and said invertebrate is not an ectoparasitic insect.
  • Another method of the invention is for inhibiting a remodelling event in an invertebrate population comprising internally contacting said invertebrate population with a compound of formula (II):
  • invertebrate remodelling event is not egg hatching and said invertebrate is not an ectoparasitic insect.
  • the invention also provides methods of inhibiting egg hatching in a non-ectoparasitic invertebrate an invertebrate population comprising contacting said invertebrate with a compound of formula (I):
  • X is selected from a covalent bond, —C(R 5 ) 2 —, —Z— or —C(R 5 ) 2 —Z—C(R 5 ) 2 —;
  • R 1 and R 1′ are independently selected from hydrogen, C 1-6 alkyl, a branched-chain C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, hydroxy, C 1-6 alkoxy, thiol, C 1-6 alkylthio, halogen, C(R 6 ) 3 , CO 2 H, CO 2 C 1-6 alkyl, SO 3 H, SO 3 C 1-6 alkyl, NH 2 , NHC 1-6 alkyl or N(C 1-6 alkyl) 2 , or R 1 and R 19 taken together are —C(R 5 ) 2 —, —C(R 5 ) 2 —C(R 5 ) 2 —, —CR 5 ⁇ CR 5 —, C(O), C(S) or NH;
  • R 2 , R 2′ , R 3 , R 3′ , R 4 and R 4′ are independently selected from hydrogen, C 1-6 alkyl, a branched-chain C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, hydroxy, C 1-6 alkoxy, thiol, C 1-6 alkylthiol, halogen, CN, C(R 6 ) 3 , CO 2 H, CO 2 C 1-6 alkyl, SO 3 H, SO 3 C 1-6 alkyl, NH 2 , NHC 1-6 alkyl or N(C 1-6 alkyl) 2 , —CH 2 CHNH(CO 2 H), NH(C 1-6 alkylene)N(C 1-6 alkyl) 2 or a 5 or 6 membered carbocyclic or heterocyclic ring; or
  • each R 5 is independently selected from hydrogen, C 1-6 alkyl, a branched-chain C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, hydroxy, C 1-6 alkoxy, thiol, C 1-6 alkylthiol, CO 2 H, CO 2 C 1-6 alkyl,
  • each R 6 is independently selected from hydrogen and halogen
  • Z is selected from a covalent bond, —NH—, —O—, —S—, —C(O)— and —C(S)—;
  • Also provided is a method of inhibiting egg hatching in a non-ectoparasitic invertebrate an invertebrate population comprising contacting said invertebrate with a compound of formula (II):
  • the non-ectoparasitic invertebrate is selected from the group consisting of nematodes, trematodes and cestodes.
  • the invertebrate is a nematode.
  • the nematode is in inhibited in its larval stage.
  • the present invention provides additional methods for identifying and selecting a chelating agent as a candidate inhibitor of invertebrate remodelling events from a collection of metal chelating agents;
  • said method comprising selecting a metal chelating agent that has at least two polar atoms capable of simultaneously coordinating with a metal ion and
  • the methods of the invention may be used for screening combinatorial libraries for rational drug design of agents that can be used as inhibitors of invertebrate remodelling and/or as pesticides in general.
  • Yet another aspect of the invention involves screening assays in which agents are identified and/or selected. Such methods involve identification or selection of a chelating agent as a candidate inhibitor of invertebrate remodelling events from a collection of metal chelating agents of formula (II):
  • Y 1 and Y 2 are independently selected form O, NR 28 , or S;
  • R 21 , R 22 , R 23 and R 24 are independently selected from hydrogen, C 1-6 alkyl, a branched-chain C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, hydroxy, C 1-6 alkoxy, thiol, C 1-6 alkylthio, halogen, CN, C(R 29 ) 3 , CO 2 H, CO 2 C 1-6 alkyl, SO 3 H, SO 3 C 1-6 alkyl, NH 2 , NHC 1-6 alkyl, N(C1-6alkyl)2 or a carbocyclic or heterocyclic ring; or
  • R 21 and R 22 or R 22 and R 23 and R 24 taken together with the carbon atoms to which they are attached form a 5 or 6 membered carbocyclic or heterocyclic ring;
  • R 25 and R 26 are independently selected from hydrogen, C 1-6 alkyl, a branched-chain C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, hydroxy, C 1-6 alkoxy, thiol, C 1-6 alkylthio, halogen, CN, C(R 29 ) 3 , CO 2 H, CO 2 C 1-6 alkyl, SO 3 H, SO 3 C 1-6 alkyl NH 2 , NHC 1-6- alkyl or N(C 1-6 alkyl) 2 ; or
  • R 25 and R 26 together with the carbon atoms to which they are attached form a 5 or 6 membered carbocyclic or heterocyclic ring;
  • R 27 is C 1-6 alkyl, a branched-chain C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, hydroxy, C 1-6 alkoxy, thiol, C 1-6 alkylthio, C(R 29 ) 2 , N(R 30 ) 2 , or a 5 or 6 membered carbocyclic ring or heterocyclic ring;
  • R 28 is hydrogen, C 1-6 alkyl, or a branched-chain C 1-6 alkyl
  • R 29 is hydrogen or halogen
  • each R 30 is independently selected from hydrogen, C 1-6 alkyl, a branched-chain C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, a 5 or 6 membered carbocyclic ring or heterocyclic ring;
  • said method comprising selecting a metal chelating agent of formula (II) that has at least two polar atoms capable of simultaneously coordinating with a metal ion and
  • the methods of the invention involve screening methods of identifying or selecting a chelating agent as a candidate inhibitor of invertebrate remodelling events from a collection of metal chelating agents of formula I:
  • X is selected from a covalent bond, —C(R 5 ) 2 —, —Z— or —C(R 5 ) 2 —Z—C(R 5 ) 2 —;
  • R 1 and R 1′ are independently selected from hydrogen, C 1-6 alkyl, a branched-chain C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, hydroxy, C 1-6 alkoxy, thiol, C 1-6 alkylthio, halogen, C(R 6 ) 3 , CO 2 H, CO 2 C 1-6 alkyl, SO 3 H, SO 3 C 1-6 alkyl, NH 2 , NHC 1-6 alkyl or N(C 1-6 alkyl) 2 , or R 1 and R 19 taken together are —C(R 5 ) 2 —, —C(R 5 ) 2 —C(R 5 ) 2 —, —CR 5 ⁇ CR 5 —, C(O), C(S) or NH;
  • R 2 , R 2′ , R 3 , R 3′ , R 4 and R 4′ are independently selected from hydrogen, C 1-6 alkyl, a branched-chain C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, hydroxy, C 1-6 alkoxy, thiol, C 1-6 alkylthiol, halogen, CN, C(R 6 ) 3 , CO 2 H, CO 2 C 1-6 alkyl, SO 3 H, SO 3 C 1-6 alkyl, NH 2 , NHC 1-6 alkyl or N(C 1-6 alkyl) 2 , —CH 2 CHNH(CO 2 H), NH(C 1-6 alkylene)N(C 1-6 alkyl) 2 or a 5 or 6 membered carbocyclic or heterocyclic ring; or
  • each R 5 is independently selected from hydrogen, C 1-6 alkyl, a branched-chain C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, hydroxy, C 1-6 alkoxy, thiol, C 1-6 alkylthiol, CO 2 H, CO 2 C 1-6 alkyl, SO 3 H, SO 3 C 1-6 alkyl, NH 2 , NHC 1-6 alkyl or N(C 1-6 alkyl) 2 ;
  • each R 6 is independently selected from hydrogen and halogen
  • Z is selected from a covalent bond, —NH—, —O—, —S—, —C(O)— and —C(S)—;
  • said method comprising selecting a metal chelating agent of formula (I) that has at least two polar atoms capable of simultaneously coordinating with a metal ion and
  • the screening assays of the invention may be combined with conventional biological assays employed to determine the efficacy of a given agent as a pesticide.
  • the screening assays above can be combined with assays designed to determine the effect on egg hatching, moulting, metamorphosis and the like as well as in vitro enzyme assays that determine the activity of one or more of the enzymes involved in one or more of the remodelling events.
  • the screening assays may be combined with assays that determine the efficacy of the compounds as inhibitors of proteases and the like.
  • FIG. 1 shows a gelatine substrate SDS-PAGE analysis of protease activity of washings obtained from various samples of hair and lice eggs (egg shell washings ESW) following staining of the gel with Coomassie blue and destaining.
  • Lane 1 shows protease activity detected in the washings obtained from unhatched lice eggs within 12 hours of hatching (sample 1) in the higher molecular weight region of the SDS gel, above 50 kDa ( FIG. 1A , lane 1).
  • a similar pattern of protease activity was detected in the washings taken from human hair samples following the removal of the louse eggs (sample 2) ( FIG. 1A , lane 2).
  • proteases were observed in the ESWs from hypochlorite treated eggs collected up to 2 hours post egg-hatching (sample 5) ( FIG. 1B ). Bands of protease activity were detected around 25-30 kDa, 50 kDa and there were a number of fainter bands detected above 50 kDa. These proteases were specifically associated with the lice eggs at the time of egg hatching and were termed egg shell washings (ESW).
  • ESW egg shell washings
  • FIG. 2 The proteases present in the louse ESWs were further characterised by their mechanistic class. Incubation with the metal chelating agents EDTA and 1,10-phenanthroline, to inhibit metalloproteases, resulted in a reduction in protease activity compared to the untreated controls ( FIGS. 2A and 2B , respectively). In contrast, there was no apparent reduction in protease activity when the ESW were incubated with the serine/cysteine protease inhibitor PMSF ( FIG. 2B ), the cysteine protease inhibitor E-64 ( FIG. 2B ) or the aspartic protease inhibitor pepstatin (data not shown).
  • the metal chelating agents EDTA and 1,10-phenanthroline to inhibit metalloproteases
  • FIG. 3 shows a two-dimensional gelatin SDS-PAGE that was used to more accurately assess the number of protease species present in the louse ESWs.
  • Each of the three main regions of protease activity in the one-dimensional gelatin SDS-PAGE ( FIG. 1B ) resolved to a number of distinct proteases present in the louse ESWs with activity in the 25-30 kDa molecular weight range resolved to at least seven distinct proteases with isoelectric points in the neutral to alkaline pH range, whereas the band of protease activity around 50 kDa resolved to at least eleven distinct protease regions with iso-electric points in the acidic to neutral pH region. At least five proteases with molecular weights above 75 kDa were also observed.
  • FIG. 4 shows the effect of 1,10-phenanthroline on egg hatching in lice. Eggs were treated 5 days post laying and then hatching observed over time.
  • FIG. 5 shows the effect of Lannate®, containing methomyl, on egg hatching in Helicoverpa eggs.
  • the ovicidal efficacy was assessed at 5 mM, 2.5 mM, 2.25 mM and 0.125 mM of methomyl.
  • FIG. 6 shows the effect of 2-acetyl-1-tetralone on egg hatching in Helicoverpa armigera eggs.
  • the ovicidal efficacy was assessed at 2 mM and 1 mM 2-acetyl-1-tetralone.
  • FIG. 7 shows the effect of 2-acetyl-1-tetralone on egg hatching in Plutella eggs.
  • the ovicidal activity was assessed at 2 mM, 1 mM, 0.5 mM and 0.1 mM.
  • FIG. 8 shows the effect of 5,5′-dimethyl-2,2′-dipyridyl on egg hatching in H. contortus eggs.
  • the ovicidal efficacy was assessed at 180 ⁇ g/mL, 18 ⁇ g/mL, 1.8 ⁇ g/mL and 0.18 ⁇ g/mL.
  • FIG. 9 shows the effect of ivermectin on egg hatching in H. contortus eggs.
  • the ovicidal efficacy was assessed at 200 ⁇ g/mL, 100 ⁇ g/mL, 50 ⁇ g/mL, 25 ⁇ g/mL and 12.5 ⁇ g/mL.
  • FIG. 10 shows the effect of 2-acetyl-1-tetralone on egg hatching in H. contortus eggs.
  • the ovicidal efficacy was assessed at 110 ⁇ g/mL and 22 ⁇ g/mL.
  • protease enzymes are involved in hydrolysing proteins in eggs, sheaths, carapaces, exoskeletons, cysts, cocoons or ootheca, weakening the structure and at least partially allowing the invertebrate to free themselves from the structure.
  • the remodelling process indirectly involves a protease enzyme, for example, a given protein or peptide may be required for the remodelling process such as a hormone that signals that the remodelling process should occur and the compositions of the invention are able to inhibit the production or processing of such a hormone.
  • the protease enzyme is a metalloprotease enzyme.
  • remodeling event refers to an event in the life cycle of an invertebrate that alters the invertebrates' immediate environment or alters the invertebrates' physical form and facilitates progression of the organism from one life stage in the life cycle to the next life stage.
  • remodelling events include egg hatching, excystment, apolysis of a cuticle or exoskeleton, ecdysis of a cuticle or exoskeleton and metamorphosis.
  • egg hatching refers to the hatching of an invertebrate from a thin membrane egg where hatching is assisted by protease enzymes.
  • Thin membrane eggs include those eggs that possess shells or cuticles comprising predominantly a protein matrix, with or without tanned proteins, are permeable to gas but essentially water impermeable, are generally non-mineralised are less than 20 mm in length and are not amenable to hatching solely by mechanical means, for example, by chewing or unassisted bursting.
  • Ecdysis refers to the shedding of an old cuticle. Ecdysis occurs after apolysis. After apolysis, moulting fluid containing inactive enzymes are secreted into the space between the epidermis and the old cuticle. The new cuticle is then formed. The enzymes in the moulting fluid are then activated and the lower regions of the old cuticle, the endocuticle and mesocuticle, are digested. The exocuticle and epicuticle of the old cuticle, which are not digested, are then shed.
  • the pest is contacted with the invention because the host of the pest has ingested or been in contact with the composition and by being in physical contact with the host, the pest either ingests or is externally contacted with the compositions of the invention.
  • the compositions and methods of the invention are employed to kill, inhibit or otherwise disrupt the life cycle stage that is exposed to the compositions of the invention. For example, where the composition is used to inhibit egg hatching, the composition is exposed directly to the invertebrate egg rather than being exposed to a different stage in the life cycle of the invertebrate.
  • Molar refractivity is a measure of the volume occupied by an atom or group and depends on temperature, the index of refraction, pressure. Molar refractivity provides an indication of size of the molecule and the polarizability of the molecule. Molar refractivity was calculated using the CMR module (Calculated Molar Refractivity) from the ClogP software program (Biobyte).
  • Log LD 50 is obtained from the observed percentage ovicidal activity by conversion using a modified logit transformation.
  • the observed percentage ovicidal activity values were transformed using the equation:
  • X is selected from a covalent bond, —C(R 5 ) 2 —, —Z— or —C(R 5 ) 2 —Z—C(R 5 ) 2 —;
  • R 2 , R 2′ , R 3 , R 3′ , R 4 and R 4′ are independently selected from hydrogen, C 1-6 alkyl, a branched-chain C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, hydroxy, C 1-6 alkoxy, thiol, C 1-6 alkylthiol, halogen, CN, C(R 6 ) 3 , CO 2 H, CO 2 C 1-6 alkyl, SO 3 H, SO 3 C 1-6 alkyl, NH 2 , NHC 1-6 alkyl or N(C 1-6 alkyl) 2 , —CH 2 CHNH(CO 2 H), NH(C 1-6 alkylene)N(C 1-6 alkyl) 2 or a 5 or 6 membered carbocyclic or heterocyclic ring; or
  • each R 5 is independently selected from hydrogen, C 1-6 alkyl, a branched-chain C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, hydroxy, C 1-6 alkoxy, thiol, CO 2 H, CO 2 C 1-6 alkyl, SO 3 H, SO 3 C 1-6 alkyl, NH 2 , NHC 1-6 alkyl or N(C 1-6 alkyl) 2 ;
  • Z is selected from a covalent bond, —NH—, —O—, —S—, —C(O)— and —C(S)—;
  • R 1 and R 1′ are independently selected from C 1-6 alkyl, a branched-chain C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, hydroxy, C 1-6 alkoxy, thiol, CO 2 H, CO 2 C 1-6 alkyl, SO 3 H, SO 3 C 1-6 alkyl, NH 2 , NHC 1-6 alkyl or N(C 1-6 alkyl) 2 , more preferably hydrogen or C 1 -C 3 alkyl, even more preferably hydrogen or methyl;
  • R 2 and R 2 are independently hydrogen or C 1-3 alkyl, more preferably hydrogen;
  • each R 5 is independently selected from hydrogen, C 1-6 alkyl, a branched-chain C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 1-6 alkoxy, C 1-6 alkylthiol or CO 2 C 1-6 alkyl, preferably hydrogen or C 1-3 alkyl, more preferably hydrogen or methyl;
  • each R 6 is independently hydrogen or fluorine, especially where each R 6 is fluorine;
  • X is a covalent bond, —CH 2 —Z—CH 2 — or Z, preferably a covalent bond
  • Z is —NH—, —O— or —S—, preferably —NH—.
  • Preferred compounds are biaryl compounds of formula (I):
  • X is selected from a covalent bond, —C(R 5 ) 2 —, —Z— or —C(R 5 ) 2 —Z—C(R 5 ) 2 —;
  • R 1 and R 19 are independently selected from hydrogen, C 1-6 alkyl, a branched-chain C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, hydroxy, C 1-6 alkoxy, thiol, C 1-6 alkylthio, halogen, C(R 6 ) 3 , CO 2 H, CO 2 C 1-6 alkyl, SO 3 H, SO 3 C 1-6 alkyl, NH 2 , NHC 1-6 alkyl or N(C 1-6 alkyl) 2 ;
  • R 2 , R 2′ , R 3 , R 3′ , R 4 and R 4′ are independently selected from hydrogen, C 1-6 alkyl, a branched-chain C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, hydroxy, C 1-6 alkoxy, thiol, C 1-6 alkylthiol, halogen, CN, C(R 6 ) 3 , CO 2 H, CO 2 C 1-6 alkyl, SO 3 H, SO 3 C 1-6 alkyl, NH 2 , NHC 1-6 alkyl or N(C 1-6 alkyl) 2 , —CH 2 CHNH(CO 2 H), NH(C 1-6 alkylene)N(C 1-6 alkyl) 2 or a 5 or 6 membered carbocyclic or heterocyclic ring; or
  • each R 6 is independently selected from hydrogen and halogen
  • Z is selected from a covalent bond, —NH—, —O—, —S—, —C(O)— and —C(S)—;
  • Preferred compounds of formula (I) include
  • the preferred metal chelating agent is a compound of formula II:
  • Preferred compounds of formula (II) have at least one of the following features:
  • Preferred compounds of formula (II) include:
  • alkyl refers to a straight-chain or branched saturated hydrocarbon group and may have a specified number of carbon atoms.
  • C 1 -C 6 as in “C 1 -C 6 alkyl” includes groups having 1, 2, 3, 4, 5 or 6 carbons in a linear or branched arrangement.
  • alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, t-butyl, n-pentyl, 2-methylbutyl, 3-methylbutyl, 4-methylbutyl, n-hexyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 5-methylpentyl, 2-ethylbutyl and 3-ethylbutyl.
  • C 1-6 alkyl as used herein also includes branched chain C 1-6 alkyl.
  • alkenyl refers to a straight-chain or branched hydrocarbon group having one or more double bonds between carbon atoms and may have a specified number of carbon atoms.
  • C 2 -C 6 as in “C 2 -C 6 alkenyl” includes groups having 2, 3, 4, 5 or 6 carbon atoms in a linear or branched arrangement.
  • suitable alkenyl groups include, but are not limited to, ethenyl, propenyl, isopropenyl, butenyl, pentenyl and hexenyl.
  • alkynyl refers to a straight-chain or branched hydrocarbon group having one or more triple bonds between carbon atoms, and may have a specified number of carbon atoms.
  • C 2 -C 6 as in “C 2 -C 6 alkynyl” includes groups having 2, 3, 4, 5 or 6 carbon atoms in a linear or branched arrangement.
  • suitable alkynyl groups include, but are not limited to, ethynyl, propynyl, butynyl, pentynyl and hexynyl.
  • halo or “halogen” refers to fluorine (fluoro), chlorine (chloro), bromine (bromo) and iodine (iodo).
  • alkyloxy or “alkoxy” as used herein represents an alkyl group as defined above attached through an oxygen bridge.
  • suitable alkyloxy groups include, but are not limited to, methoxy, ethoxy, n-propyloxy, i-propyloxy, n-butyloxy, i-butyloxy, t-butyloxy, n-pentyloxy and n-hexyloxy.
  • alkylthio represents an alkyl group as defined above attached through a sulfur bridge.
  • suitable alkylthio groups include, but are not limited to, methylthio, ethylthio, propylthio, i-propylthio, butylthio, i-butylthio, t-butylthio, pentylthio, hexylthio.
  • carbocyclic ring refers to a 3 to 10 membered ring or fused ring system, in which all of the atoms that form the ring are carbon atoms.
  • the C 3-10 carbocyclic ring may be saturated, unsaturated or aromatic.
  • suitable carbocyclic rings include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, phenyl, naphthyl and tetrahydronaphthyl.
  • heterocyclic ring refers to a 3 to 10 membered ring or fused ring system in which at least one of the atoms that form the ring is a heteroatom.
  • the heteroatom is selected from nitrogen, oxygen, sulfur and phosphorus.
  • the C 3-10 heterocyclic ring may be saturated, unsaturated or aromatic.
  • heterocyclic rings include, but are not limited to, benzoimidazolyl, benzofuranyl, benzofurazanyl, benzopyrazolyl, benzotriazolyl, benzothiophenyl, benzoxazolyl, carbazolyl, carbolinyl, cinnolinyl, furanyl, imidazoyl, indolinyl, indolyl, indolazinyl, indazolyl, isobenzofuranyl, isoindolyl, isoquinolyl, isothiazolyl, isoxazolyl, naphthpyridinyl, oxadiazolyl, oxazolyl, oxazoline, isoxazoline, oxetanyl, pyranyl, pyrazinyl, pyrazolyl, pyridazinyl, pyridopyridinyl, pyridazinyl, pyridyl,
  • aryl is intended to mean any stable, monocyclic or bicyclic carbon ring of up to 6 atoms in each ring, wherein at least one ring is aromatic. Examples of such aryl groups include, but are not limited to, phenyl, naphthyl and tetrahydronaphthyl.
  • heteroaryl represents a stable monocyclic or bicyclic ring of up to 6 atoms in each ring, wherein at least one ring is aromatic and at least one ring contains from 1 to 4 heteroatoms selected from the group consisting of O, N and S.
  • Heteroaryl groups within the scope of this definition include, but are not limited to, acridinyl, carbazolyl, cinnolinyl, quinoxalinyl, pyrrazolyl, indolyl, benzotriazolyl, furanyl, thienyl, benzothienyl, benzofuranyl, quinolinyl, isoquinolinyl, oxazolyl, isoxazolyl, indolyl, pyrazinyl, pyridazinyl, pyridinyl, pyrimidinyl, pyrrolyl, tetrahydroquinoline.
  • the compounds of the invention may be in the form of pharmaceutically, veterinary or agriculturally acceptable salts.
  • suitable pharmaceutically acceptable salts include, but are not limited to, salts of pharmaceutically acceptable inorganic acids such as hydrochloric, sulphuric, phosphoric, nitric, carbonic, boric, sulfamic, and hydrobromic acids, or salts of pharmaceutically acceptable organic acids such as acetic, propionic, butyric, tartaric, maleic, hydroxymaleic, fumaric, maleic, citric, lactic, mucic, gluconic, benzoic, succinic, oxalic, phenylacetic, methanesulphonic, toluenesulphonic, benezenesulphonic, salicyclic sulphanilic, aspartic, glutamic, edetic, stearic, palmitic, oleic, lauric, pantothenic, tannic, ascorbic and valeric acids.
  • Base salts include, but are not limited to, those formed with pharmaceutically acceptable cations, such as sodium, potassium, lithium, calcium, magnesium, ammonium and alkylammonium.
  • Basic nitrogen-containing groups may be quarternised with such agents as lower alkyl halide, such as methyl, ethyl, propyl, and butyl chlorides, bromides and iodides; dialkyl sulfates like dimethyl and diethyl sulfate; and others.
  • lower alkyl halide such as methyl, ethyl, propyl, and butyl chlorides, bromides and iodides
  • dialkyl sulfates like dimethyl and diethyl sulfate; and others.
  • the invention thus also relates to compounds in substantially pure isomeric form at one or more asymmetric centres e.g., greater than about 90% ee, such as about 95% or 97% ee or greater than 99% ee, as well as mixtures, including racemic mixtures, thereof.
  • Such isomers may be prepared by asymmetric synthesis, for example using chiral intermediates, or by chiral resolution.
  • metal chelating agents and metalloprotease inhibitors useful in the present invention can be obtained commercially from specialty chemical companies. Those not commercially available can be synthesised from commercially available starting materials using reactions known to those skilled in the art.
  • substituted 2,2′-bipyridyls and 1,10-phenanthrolines may be obtained from suitable halogenated 2,2′-bipyridyls or 1,10-phenanthrolines.
  • 2,2′-bipyridin-6,6′-dicarboxylic acid may be obtained from 6,6′-dibromo-2,2′-dipyridyl by halogen-metal exchange with butyl lithium, treatment with dry ice and acidification [Buhleier et. al., Chem. Ber., 1978, 111: 200-204].
  • Monosubstitution of a bipyridyl for example with CH 2 CHNH 2 (CO 2 H) at the 6 position, can be obtained by treatment of 6-methyl-2,2′-bipyridyl with N-bromosuccinimide followed by alkylation with N-protected-glycine ester.
  • the protecting groups can then be removed by acid hydrolysis, (Imperiali B. and Fisher S. L., J. Org. Chem., 1992, 57: 757-759).
  • 2,2′-Dipyridyls can undergo nucleophilic substitution at the C6 and C4 positions to introduce substituents. This reaction is more favorable when a halogenated dipyridyl is used as the starting material.
  • a halogenated dipyridyl is used as the starting material.
  • an amine may be introduced at C6 and/or C6′ by using 6-mono or di-halogenated 2,2′-dipyridyl and reacting this starting material with ammonia.
  • Bipyridyl-sulfonic acids can be prepared from 2,2′-bipyridyl by heating with either oleum (a solution of sulfur trioxide in concentrated sulfuric acid) or mercury (II) sulfate/concentrated sulfuric acid at 300° C.
  • Unsymmetrically substituted bipyridyls can be obtained from symmetrical bipyridyls, for example, 6′-methyl-2,2′-bipyridyl-6-carboxylic acid can be prepared from 6,6′-dimethyl-2,2′-bipyridyl by oxidation with selenium dioxide followed by treatment with silver nitrate (Al-Saya et. al., European J. Org. Chem., 2004, 173-182).
  • insects that are inhibited from undergoing remodelling events in the present invention are pests that internally or externally infest humans or animals, infest plants or infest property or a particular environment.
  • pests that internally infest humans or animals include, but are not limited to, nematodes, trematodes and cestodes
  • pests that externally infest humans or animals include, but are not limited to, lice, fleas, mites and scabies
  • pests that infest plants include, but are not limited to, moths, beetles, caterpillars butterflies and nematodes
  • pests that damage property include, but are not limited to, termites and pests that infest an environment include, but are not limited to, arachnids, cockroaches, centipedes, fleas and mites.
  • a method of treating or preventing a pest infestation of a host or environment comprising applying or administering to the host or environment an effective amount of at least one metal chelating agent, wherein the metal chelating agent has at least two polar atoms capable of simultaneously coordinating with a metal ion and
  • the host treated by the methods of the invention may be selected from, but is not limited to, the group consisting of humans, sheep, cattle, horses, pigs, poultry, dogs and cats.
  • the methods of treatment or prevention of the present invention may be applicable to plants and or other breeding, feeding or habitation sites of pests.
  • Plants treated by the methods of the invention are preferably selected from, but are not limited to, the group consisting of cotton, oil seed or cereal grain crops such as canola, forestry crops such as trees, specimen plants such as trees, ornamental plants such as shrubs, flowers such as chrysanthemum, michaelmas daisy, geraniums and pinks, fruit trees such as apples, pears, plums, kiwifruit and citrus varieties for example, lemons, oranges, limes and grapefruit, cereal crops such as maize and sweet corn, vine crops such as grapes, root crops, pasture plants such as red and white clover, lucerne and lupins, and vegetables such as brassica crops, for example, broccoli and cauliflower, cabbage, tomatoes, zucchini, leeks, lettuce and beans as well as pulses such as navy beans, soybeans, mungbeans, pigeon pease and chickpeas and vine crops such as grapes.
  • the environment to be treated by the methods of the present invention includes the surroundings of an animal, human or plant that is or may become infested with a pest and includes but is not limited to soils surrounding plants or houses, gardens, lawns, kennels, barns or animal enclosures, carpets, clothing, bed linen and beds and the breeding sites of pests.
  • the environment also includes property that may be damaged by a pest, for example buildings, furniture and wooden products that may be damaged or destroyed by termites.
  • Preferred pests that undergo remodelling events and may be controlled by the methods described include but are not limited to a species from a class, subclass or an order selected from the phylum Platheminthes such as the classes Cestoda and Trematoda, from the phylum Nematoda such as the classes Adenophoria or Secernentia, from the phylum Arthropoda such as the classes Crustacea, Arachnida, Insecta and Acarina.
  • Tissue flukes are species which infect the bile ducts, lungs, or other biological tissues which includes the lung fluke, Paragonimus westermani , and the liver flukes, Clonorchis sinensis, Fasciola hepatica and Fasciola gigantica .
  • the other group are known as blood flukes, and inhabit the blood in some stages of their life cycle. Blood flukes include various species of the genus Schistosoma.
  • Nematodes commonly parasitic on humans include whipworms, hookworms, pinworms, ascarids, and filarids.
  • Within the nematode phylum is the class Adenophoria, and the subclass Enoplia that include the roundworms.
  • Most nematodes in this subclass are free-living, but the group also includes the order Trichiurida, which includes the parasitic whipworms and trichina worms.
  • nematodes Also within the nematodes are the Secernentea, subclass Rhabditia that is mostly comprised of parasitic nematodes, though there are some free-living species as well.
  • An important order is the Ascaridida, which includes worms that infect many land mammals and marine mammals. Important families within this order include Ascarididae, which includes the giant intestinal roundworm and related species and Toxocaridae, which includes parasites of canids, felids, and raccoons, but which can aberrantly parasitize humans and cause visceral larval migrans.
  • Strongylida which includes the genus Metastrongylus a nematode of the family Metastrongylidae, usually found as lungworms in pigs and sometimes causing verminous bronchitis.
  • nematode parasites In addition to the nematode parasites of mammalian hosts there are several groups of plant parasitic nematodes that can cause severe crop losses. The most common genera are Aphelenchoides (foliar nematodes), Meloidogyne (root-knot nematodes), Heterodera, Globodera (cyst nematodes), such as the potato nematode, Nacobhus, Pratylenchus (lesion nemtodes), Ditylenchus Xiphinema, Longidorus, Trichodorus .
  • Aphelenchoides foliar nematodes
  • Meloidogyne root-knot nematodes
  • Heterodera Heterodera
  • Globodera cyst nematodes
  • potato nematode such as the potato nematode, Nacobhus, Pratylenchus (lesion nemtodes), Dity
  • nematode species cause histological damage to roots, including the formation of visible galls ( Meloidgyne ) which are useful characters for their diagnostic in the field.
  • Some nematode species transmit plant viruses through their feeding activity in roots.
  • One of these nematodes is Xiphinema index , vector of GFLV (Grapevine Fanleaf Virus), an important disease of grapes.
  • Other nematodes attach bark and forest trees. The most important representative of this group is Bursaphelenchus xylophilus , the pine wood nematode, present in Asia and America and recently discovered in Europe.
  • orders include: Lepidoptera, Hemiptera, Orthoptera, Psocoptera, Hymenoptera, Isoptera, Coleoptera, Dictyoptera, Thysanoptera, Homoptera , Diptera, Siphonaptera and Phthiraptera that comprises the Anoplura and Mallophaga.
  • Suitable pests that may be controlled using the methods of the present invention include:
  • Helicoverpa spp. such as Helicoverpa armigera, Helicoverpa Zea and Helicoverpa punctigera (Budworms), Crocidolomia pavonana (Cabbage cluster caterpillar), Pieris rapae (Cabbage white butterfly), Phthorimaea operculella (Potatoe moth), Chrsyodexis spp.
  • Especially preferred pests that infest domestic animals include Bovicola ovis (Sheep louse), Bovicola bovis, Haematopinus eurysternus (short-nosed cattle louse), Linognathus vituli (long nosed cattle louse), Solenopotes capillatus (tubercule-bearing louse), Sarcoptes scabiei canis (mange), Sarcoptes scabiei suis, Sarcoptes scabiei bovis, Psoroptes ovis, Haemonchus contortus, H. placei, Teladorsagia circumcincta, Trichostrongylus colubriformis , and Cooperia spp.
  • Especially preferred pests that infest humans include Pthirus pubis, Pediculus humanus capitus, Pediculus humanus humanus, Sarcoptes scabiei var. humani and Dermatophgoides spp.
  • the pest which is prevented from undergoing a remodelling event by the present invention is selected from the group consisting of louse, flea, tick, fly, mite and other biting or blood-sucking pest eggs.
  • the pest egg is a louse egg, more preferably head louse egg.
  • Lice are a parasite that feed on animal skin and blood and they deposit their digestive juices and faecal material into the skin. These materials, as well as the puncture wound itself, cause skin irritation and lesions from the resulting scratching, and can cause a serious infection with ganglionic inflammation. Lice are also vectors of certain diseases, such as exanthematic or epidemic typhus and recurrent fever.
  • the adult female louse has a life span of about one month and can lay up to ten eggs a day.
  • Lice that infect humans may include the species of crab louse ( Pthirus pubis ) and the separate species Pediculus humanus which is composed of two subspecies, Pediculus humanus capitis or head lice and Pediculus humanus humanus or clothing lice (Busvine, Antenna, 1993, 17: 196-201).
  • the above subspecies of lice are closely related and are known to successfully interbreed in the laboratory situation (Busvine, Cutaneous Infestations and Insect Bites, 1985, 163-174).
  • the head louse Pediculus humanus var. capitis is a host-specific ectoparasite that lives exclusively on human heads and feeds via sucking blood from the scalp. Following a blood meal, mature adult female lice will lay up to 10 eggs close to the scalp over a 24 hour period. The eggs are attached firmly to the hair shaft via a glue. Seven to ten days post laying depending on temperature and humidity, the eggs will hatch and the newly emerged nymphs begin to feed. The nymphs progress through three moults (1 st instar, 2 nd instar, 3 rd instar) with each moult taking between 3-5 days to complete. Following the final moult the adult male or female emerges with mating taking place as early as two days later.
  • the pest which is prevented from undergoing a remodelling event by the present invention is one that infests a plant host including the plant's roots.
  • the pest is a budworm egg, a caterpillar egg, a butterfly, a moth or a root nematode.
  • valuable crop plants such as cotton, oil seed crops such as canola, ornamental plants, flowers, fruit trees, cereal crops, vine crops, root crops, pasture crops, tobacco, pulses and vegetables, especially Brassica crops such as cauliflower and broccoli, cotton, maize, sweetcorn, tomatoes, tobacco and pulses such as soybeans, navy beans, mungbeans, pigeon peas and chickpeas.
  • the diamondback moth Plutella xylostella ) larvae feed on all plants in the Brassica /cruciferae family, including canola and mustard, vegetable crops such as broccoli, cauliflower and cabbage and also on several greenhouse plants. Normally the diamondback moth takes about 32 days to develop from egg to adult. However, depending on food and weather conditions, a generation may take from 21 to 51 days to complete.
  • Adult female moths lay an average of 160 eggs over a lifespan of about 16 days. A female will lay eggs at night and will lay the largest number of eggs the first night after emergence from the pupa. The eggs are small, spherical or oval and yellowish-white and are glued to the upper or lower surfaces of a leaf either singly or in groups of two or three.
  • the eggs are usually laid along the veins of the leaf where the leaf surface is uneven.
  • the eggs hatch in about five to six days. After hatching, the larvae burrow into the leaf and begin eating the leaf tissue internally. After about a week, the larvae exit from the leaf and feed externally.
  • the larvae moult three times over 10 to 21 days and at maturity are about 12 mm long.
  • the larvae pupate in delicate, open-mesh cocoons attached to the leaves and the pupal stage lasts from 5 to 15 days.
  • Budworms such as corn ear worm, tomato grub, tobacco budworm and cotton Bollworm are serious pests in a number of crops such as sunflowers, zucchini, beans, peppers, alfalfa, potatoes, leeks, cotton, maize, plums, citrus plants, tomatoes, tobacco and lettuce, and flowers such as geraniums and pinks.
  • Budworms occur in many regions of the world and in temperate climates may have 2-3 generations per season with pupae overwintering in the soil. In tropical regions, the budworms may continue to be active year round. Eggs are small ( ⁇ 0.5 mm in diameter) and dome shaped with a slightly flattened bottom. Eggs are usually laid singularly near buds or flowering parts or on leaves. An adult may lay 500-3000 eggs.
  • the eggs hatch after only three days at 25° C. or longer at cooler temperatures, for example, 9 days at 17° C.
  • the larval feeding period is about 19 to 26 days under favourable temperature and feeding conditions and when fully developed the larvae move to the soil to pupate.
  • the pupal period generally lasts from 8 to 21 days although diapausing pupae can overwinter in soil in temperate regions.
  • the pest that is prevented from undergoing a remodelling event by the present invention is one that internally infests a human or animal.
  • the pest is a nematode, a trematode or a cestode.
  • Nematodes (roundworms), trematodes and cestodes are flat worms and may cause significant damage to humans or agriculturally important animals such as sheep, cows, pigs and goats.
  • Haemonchus contortus an intestinal parasite that infests sheep and goats, and adult male and female worms live in the abomasum or the true stomach of ruminant animals.
  • the female worms deposit 5,000 to 10,000 eggs per day which are passed out of the host with the faeces. After hatching the first and second stage juveniles feed on bacteria.
  • the third stage juveniles retain a cuticle as a sheath and the third stage juvenile is ingested by the host while grazing.
  • the young sheathed worms pass into the host and exsheath before entering the abomasum. In the abomasum the exsheathed young worms burrow into the mucosa and feed on blood.
  • the methods and compositions are to treat or prevent external infestation of a human or animal by a pest or parasite that undergoes remodelling events, such as lice, fleas, mites or ticks, by inhibiting these remodelling events.
  • the inhibition of remodelling events has the advantage of interrupting the life cycles and/or breeding cycles of the pest or parasite thereby controlling infestation.
  • the methods and compositions are to treat or prevent internal infestation of a human or animal by a pest or parasite that undergoes remodelling events, such as nematodes and trematodes, by inhibiting transition from one stage of the life cycle of the pest or parasite to the next.
  • the inhibition of remodelling events has the advantage of interrupting the life cycles and breeding cycles of the pest or parasite at a number of different points thereby controlling infestation.
  • the methods and compositions are to treat or prevent infestation of an environment with a pest or parasite by inhibiting remodelling events of the pest or parasite.
  • the eggs of pests or parasites may be laid in soil around a plant, in carpet or curtains in a house (eg: flea eggs), linen or mattresses of bedding (eg: dust mite eggs or bed bug eggs) or on or in the vicinity of wooden structures such as buildings or other wooden products (eg: termite eggs).
  • the hatching of the eggs allows reinfestation of humans, animals or plants in the environment or damage to products in the environment.
  • the inhibition of remodelling events has the advantage of interrupting the breeding cycles of the pest or parasite thereby controlling infestation.
  • the prevention of reinfestation results in a reduction of the number of applications of pesticides required to control an infestation.
  • the methods and compositions of the invention are to treat or prevent larval infestation of plants by inhibiting remodelling events.
  • the present applicants have identified metal chelating agents as effective agents for inhibiting remodelling events that affect both eggs and larvae that feed on commercially valuable plants.
  • the use of metal chelating agents for inhibiting remodelling events has the advantage of inhibiting breeding cycles of organisms that produce larvae that feed on commercially valuable plants thereby controlling pest infestation of the commercially valuable plants.
  • metalloprotease as used herein is taken to refer to a protease involved in invertebrate remodelling events during one or more stages of a pest species development, wherein the protease has an active metal ion that acts as a catalyst.
  • the metalloprotease contains a zinc ion that participates in catalysis by polarizing a water molecule to attack a substrate-peptide bond. More preferably, the metalloprotease is sensitive to metal chelating agents that are capable of either directly or indirectly blocking their activity.
  • the metalloprotease may be involved in inducing egg hatching by acting on the operculum of an egg to facilitate egg hatching or may reduce the strength of the egg shell allowing the nymph or larvae to break out of the shell during hatching.
  • the metalloprotease may also be involved in facilitating the change from one larval or immature stage to a subsequent stage and also to the adult or mature form.
  • the metalloprotease may be directly or indirectly involved in the remodelling events. Suitable metalloproteases involved in remodelling events can include endoproteases (enzymes that cleave within the peptide chain) and exoproteases (enzymes that cleave amino acid(s) from the termini of peptides).
  • Exoproteases can further be categorised as carboxyproteases (which cleave amino acid(s) from the C terminus) or aminopeptidase (which cleave amino acids from the N terminus).
  • Metallo-carboxyproteases require a bivalent cation (usually Zn 2+ ) for activity, while aminopeptidases are generally classified according to their dependence on metal ions (Zn 2+ or Mg 2+ ). They exist in both free and membrane-bound forms and favour activity at high (8-10) pH.
  • One method of detecting metalloproteases associated with egg hatching can involve collecting either the fluid surrounding the developing embryo at the time of egg hatching or by washing the empty egg shells shortly after egg hatching and analyzing the sample for the presence of proteases using gelatine substrate SDS-PAGE analysis. Having shown the presence of proteolytic activity from the sample it is then possible to incubate the sample in the presence of a metalloprotease inhibitor that has been identified as having the required arrangement of polar atoms and clogP values and/or molar refractivity and in some embodiments, a preference for chelating with zinc ions, and then reanalyze the treated sample to determine if the activity of the proteases extracted from the egg have been inhibited.
  • metalloprotease(s) obtained from the hatched egg it is then possible to expose unhatched eggs, for example, to the same inhibitor and assess whether inhibition of egg hatching occurs. Similar approaches can be made to determine metal chelating agents suitable for inhibiting other remodelling events such as apolysis, ecdysis exsheathment or metamorphosis. For example, fluid may be obtained from invertebrates undergoing apolysis, ecdysis or metamorphosis and the presence of proteases detected as described above. Suitable metal chelating agents may then be determined. Metalloproteases involved in egg hatching may also be identified by identification of a gene encoding a metalloprotease, silencing that gene and showing that the egg is unable to hatch by methods known to those skilled in the art.
  • inhibiting remodelling events is taken to mean the inhibition of protease enzymes involved in remodelling events that involve encasements of invertebrate multi-cellular organisms, for example, eggs, sheaths, carapaces, exoskeletons, cysts, cocoons or ootheca.
  • a particular life cycle stage of invertebrate pest is exposed to a metal chelating agent that is capable of preventing a remodelling event when compared to the same life cycle stage that is untreated.
  • this remodelling event may be characterised by the hatchflap or operculum of an egg opening and shortly thereafter the emergence of a larvae or nymph.
  • the metal chelating agent is a compound capable of inhibiting remodelling events when it is applied to a stage of the pests life cycle at any time between laying and throughout adults life.
  • the remodelling event preferably takes place in a pest present on, but not limited to, a host organism, such as on the skin, hair, coat or fleece of an animal or skin or hair such as head hair of a human.
  • the remodelling event takes place in a pest present on host plants or in the roots of plants including cereal crops, fruit trees, cotton, oil seed crops, ornamental plants, flowers, vine crops, root crops, pasture plants and vegetables.
  • the remodelling event takes place in a pest that is present in an environment or breeding site, such as, but not limited to, houses and buildings, enclosures for domestic and farming animals, carpets, bedding such as sheets and blankets, curtains and furniture.
  • the remodelling event may take place in a pest that is inside a host, such as, but not limited to, humans, domestic and farming animals.
  • the pest may be exposed to a metal chelating agent by any suitable means.
  • suitable means may vary widely, depending upon whether the chelating agent is to be applied to a host, such as a plant or applied or administered to an animal including a human, or applied to various environments of other breeding sites, and depending on the nature and type of pest targeted.
  • Suitable means for exposing the pest present on animals to metal chelating agents include, but are not limited to, direct topical application, such as by dipping or spraying, implants, delayed release formulations or devices, or orally.
  • formulations suitable for topical application include but are not limited to sprays, aerosols, shampoos, mousses, creams and lotions
  • formulations suitable for internal application include but are not limited to tablets, capsules or liquid formulations.
  • parenteral administration by injection may be the most suitable means of treatment for humans or animals.
  • suitable means include but are not limited to sprays, dusts including wettable powders, wettable granules and suspension concentrates, pellets, liquids including micro-encapsulations and aerosols.
  • the method of the invention also encompasses the concurrent or successive use of two or more metal chelating agents or the use of one or more metal chelating agents in conjunction concurrently or successively with other known agents that control pests.
  • the methods and compositions may include other pesticides that control hatching, larvae, nymphs or adult pests.
  • suitable pesticides which may be used in conjunction, either simultaneously, separately or sequentially, with the metal chelating agents of the present invention include macrocyclic lactones such as spinosad, botanical insecticides, carbamate insecticides, dessicant insecticides, dintrophenol insecticides, fluorine insecticides, formamidine insecticides such as armitraz, fumigant insecticides, inorganic insecticides, insect growth regulators, (including chitin synthesis inhibitors, juvenile hormone mimics, juvenile hormones, moulting hormone agonists, moulting hormone antagonists, moulting hormones, moulting inhibitors), nicotinoid insecticides, organochlorine insecticides, organophosphorus insecticides, heterocyclic organothiophosphate insecticides, phenyl organothiophosphate insecticides, phosphonate
  • Trematode and cestode infections can also be treated with Praziquantel.
  • the metal chelating agent may be applied to the hair or skin of a host when the host is a human or animal, preferably in a region that is infested with a pest.
  • the infestation may be due to pests selected from the group consisting of lice, fleas, ticks, flies, mites and other biting or blood-sucking pests, and combinations thereof.
  • the metal chelating agent may be applied topically in the form of ointments, aqueous compositions including solutions and suspensions, creams, lotions, aerosol sprays or dusting powders.
  • the metal chelating agent may be applied or administered internally, for example, in the form of a tablet, capsule or ingestable liquid formulation.
  • the pest infestation is preferably due to pests selected from, caterpillars, butterflies, moths or nematodes.
  • the metal chelating agent may be applied topically, for example, in the form of a spray or dust.
  • the metal chelating agent may be applied in a formulation such as a spray, fumigant or dust.
  • the term “effective amount” means a concentration of at least one metal chelating agent sufficient to provide treatment or prevention of a pest infestation in a host or in an environment.
  • the effective amount of a metal chelating agent used in the methods of the present invention may vary depending on the host and the type and level of infestation.
  • the metal chelating agent is applied to the scalp of a person suffering from head lice infestation and are left on the treated person for a period of time to prevent hatching of the louse eggs. Preferably the period of time is between 5 and 15 minutes.
  • the metal chelating agent is preferably used at a concentration of between about 0.0001 mM to 1M, preferably 0.01 mM and 100 mM, more preferably in the range of 0.1 mM and 100 mM.
  • the effective amount depends on the metal chelating agent used. However, some dipyridyl compounds may suitably be applied in the range of 5 mM to 100 mM, especially at a level of about 50 mM. Suitable amounts of compounds of formula (II), such as tetralone compounds, may be applied at a level in the range of 0.5 mM to 100 mM, especially 1 mM to 50 mM.
  • metal chelating agent Since a significant number of mammalian proteases require zinc for their activity and may be affected by metal chelating agents, it would be necessary to ensure that the metal chelating agent was used in a safe and effective amount and is preferably specifically targeted to a specific remodelling event, such as egg hatching, apolysis, ecdysis, exsheathment or metamorphosis.
  • the metal chelating agent is applied to a commercially valuable plant to prevent remodelling events occurring in a pest that are involved in, for example, egg hatching or moulting.
  • the metal chelating agent may be applied directly or indirectly to pests which are present in the ground or on the leaves, buds, stems, flowers or fruit of a plant by spray application, brushing on or dusting.
  • Suitable compositions include emulsifiable concentrates, directly sprayable or dilutable solutions, dilute emulsions, wettable powders, soluble powders, dusts or granules.
  • the metal chelating agent is preferably used at a concentration of between about 0.0001 mM to 1M, preferably 0.01 mM and 100 mM, more preferably in the range of 0.1 mM and 30 mM.
  • the effective amount depends on the metal chelating agent used. However, some dipyridyl compounds may suitably be applied in the range of 5 mM to 15 mM, especially at a level of about 10 mM.
  • Suitable amounts of compounds of formula (II) include, but are not limited to, the range of 0.1 mM to 20 mM, especially 1.0 mM to 15 mM.
  • the host treated by the methods of the invention may be selected from, but is not limited to, the group consisting of humans, sheep, cattle, horses, pigs, poultry, dogs and cats.
  • the methods of treatment or prevention of the present invention may be applicable to plants and or other breeding sites of pests.
  • Plants or their roots treated by the methods of the invention are preferably selected from the group consisting of cotton, oil seed crops such as canola, ornamental plants such as shrubs, flowers such as chrysanthemum, michaelmas daisy, geraniums and pinks, fruit trees such as apples, pears, plums, kiwifruit, currants and citrus varieties for example, lemons, oranges, limes and grapefruit, cereal crops such as maize and sweetcorn, vine crops such as grapes, root crops, pasture plants such as red and white clover, lucerne and lupins, and vegetables such as brassica crops, for example, broccoli and cauliflower, cabbage, tomatoes, zucchini, leeks, lettuce and beans as well as pulses such as navy beans, soybeans, mungbeans, pigeon peas and chickpeas.
  • ornamental plants such as shrubs
  • flowers such as chrysanthemum, michaelmas daisy, geraniums and pinks
  • fruit trees such
  • compositions of the present invention may be formulated as solutions and emulsions.
  • Suitable excipients such as emulsifiers, surfactants, stabilizers, dyes, penetration enhancers and anti-oxidants may also be present in the compositions.
  • Suitable carriers that may be added in the compositions can include, water, salt solutions, alcohols, polyethylene glycols, gelatine, lactose, magnesium sterate and silicic acid.
  • the compositions may include sterile and non-sterile aqueous solutions.
  • the compositions are in a soluble form and the metal chelating agent is diluted in a soluble sterile buffered saline or water solution.
  • compositions can also be formulated as suspensions in aqueous, non-aqueous or mixed media.
  • Aqueous suspensions may further contain substances that increase the viscosity of the suspension and may also contain stabilizers.
  • the solutions may also contain buffers, diluents and other suitable additives.
  • the compositions can include other adjunct components that are compatible with the activity of the metal chelating agent.
  • the compositions of the present invention may be formulated and used as foams, emulsions, microemulsions, shampoos, mousses, creams and jellies. The formulations of the above compositions described would be known to those skilled in the field of pesticides.
  • the active ingredients according to the invention can be used for inhibiting remodelling events that occur in pests on plants or in their roots, mainly on crops of useful plants and ornamentals in agriculture, in horticulture and in silviculture, or on parts of such plants, such as fruits, flowers, foliage, stalks, tubers or roots, and in some cases even parts of plants which are formed at a later point in time are afforded protection against these pests.
  • the active ingredient is employed together with at least one of the auxiliaries conventionally used in the art of formulation, such as extenders, eg solvents or solid carriers, or such as surface-active compounds (surfactants).
  • suitable solvents are: non-hydrogenated or partially hydrogenated aromatic hydrocarbons, preferably the fractions C 8 -C 12 of alkylbenzenes, such as xylene mixtures, alkylated naphthalenes or tetrahydronaphthalene, aliphatic or cycloaliphatic hydrocarbons such as paraffins or cyclohexane, alcohols such as methanol, ethanol, propanol or butanol, glycols and their ethers and esters such as propylene glycol, dipropylene glycol ether, hexylene glycol, ethylene glycol, diethoxy glycol, ethylene glycol monomethyl ether or ethylene glycol monoethyl ether, ketones such as cyclohexanone, isophorone or diacetone alcohol, strongly polar solvents such as N-methylpyrrolid-2-one, N-methyl-pyrrolidine, dimethyl sulfoxide or N,N-dimethylformamide
  • Solid carriers which are used for example for dusts and dispersible powders are, as a rule, ground natural minerals, such as calcite, talc, kaolin, montmorillonite or attapulgite.
  • ground natural minerals such as calcite, talc, kaolin, montmorillonite or attapulgite.
  • highly-disperse silicas or highly-disperse absorptive polymers are also possible to add highly-disperse silicas or highly-disperse absorptive polymers.
  • Suitable particulate adsorptive carriers for granules are porous types, such as pumice, brick grit, sepiolite or bentonite, and suitable non-sorptive carrier materials are calcite or sand.
  • a large number of granulated materials of inorganic or organic nature can be used, in particular dolomite or comminuted plant residues.
  • Suitable surface-active compounds are, depending on the nature of the active ingredient to be formulated, non-ionic, cationic and/or anionic surfactants or surfactant mixtures which have good emulsifying, dispersing and wetting properties.
  • the surfactants listed below are only to be considered as examples; many more surfactants conventionally used in the art of formulation and suitable in accordance with the invention are described in the relevant literature.
  • Suitable non-ionic surfactants are primarily polyglycol ether derivatives of aliphatic or cycloaliphatic alcohols, of saturated or unsaturated fatty acids and alkylphenols which can contain 3 to 30 glycol ether groups and 8 to 20 carbon atoms in the (aliphatic) hydrocarbon radical and 6 to 18 carbon atoms in the alkyl radical of the alkylphenols. Also suitable are water-soluble polyethylene oxide adducts with polypropylene glycol, ethylenediaminopolypropylene glycol and alkylpolypropylene glycol having 1 to 10 carbons in the alkyl chain and 20 to 250 ethylene glycol ether and 10 to 100 propylene glycol ether groups.
  • the above-mentioned compounds normally contain 1 to 5 ethylene glycol units per propylene glycol unit.
  • examples which may be mentioned are nonylphenylpolyethoxyethanols, castor oil polyglycol ethers, polypropylene/polyethylene oxide adducts, tributylphenoxypolyethoxyethanol, polyethylene glycol and octylphenoxypolyethoxyethanol.
  • fatty acid esters of polyoxyethylene sorbitan such as polyoxyethylene sorbitan trioleate.
  • the cationic surfactants are mainly quaternary ammonium salts which have, as substituents, at least one alkyl radical of 8 to 22 carbon atoms and, as further substituents, lower alkyl, benzyl or lower hydroxyalkyl radicals which may be halogenated.
  • the salts are preferably in the form of halides, methylsulfates or ethylsulfates. Examples are stearyltrimethylammonium chloride and benzyldi(2-chloroethyl)ethylammonium bromide.
  • Suitable anionic surfactants can be both water-soluble soaps and water-soluble synthetic surface-active compounds.
  • Soaps which are suitable are the alkali metal salts, alkaline earth metal salts and unsubstituted or substituted ammonium salts of higher fatty acids (C 10 -C 22 ), such as the sodium or potassium salts of oleic or stearic acid, or of natural fatty acid mixtures which can be obtained, for example, from coconut or tall oil; or fatty acid methyltaurinates.
  • synthetic surfactants in particular fatty sulfonates, fatty sulfates, sulfonated benzimidazole derivatives or alkylarylsulfonates, are used more frequently.
  • the fatty sulfonates and fatty sulfates exist as alkali metal salts, alkaline earth metal salts or unsubstituted or substituted ammonium salts and generally have an alkyl radical of 8 to 22 carbon atoms, alkyl also including the alkyl moiety of acyl radicals.
  • Examples of fatty sulfonates and fatty sulfates include the sodium or calcium salt of lignosulfonic acid, of the dodecylsulfuric ester or of a fatty alcohol sulfate mixture prepared with natural fatty acids. This group also includes the salts of the sulfuric esters and sulfonic acids of fatty alcohol/ethylene oxide adducts.
  • the sulfonated benzimidazole derivatives preferably contain 2 sulfo groups and one fatty acid radical having approximately 8 to 22 carbon atoms.
  • alkylarylsulfonates are the sodium, calcium or triethanolammonium salts of dodecylbenzenesulfonic acid, of dibutylnaphthalenesulfonic acid or of a naphthalenesulfonic acid/formaldehyde condensate.
  • corresponding phosphates such as salts of the phosphoric ester of a p-nonylphenol(4-14)ethylene oxide adduct, or phospholipids.
  • the composition comprises a metal chelating agent at a concentration of about 0.0001 mM to 1M, preferably between 0.1 mM to 100 mM, more preferably in the range of 0.1 mM to 50 mM.
  • Compositions containing some metal chelating agents, for example, the compounds of formula I may preferably contain between 5 and 50 mM of compound, especially at a level of about 30 mM.
  • Compositions containing compounds of formula (II) may preferably contain between 0.1 mM to 100 mM, especially 1.0 mM to 50 mM.
  • a compound which inhibits egg hatching remodelling events in a pest may be identified using a method comprising assessing the clogP value and/or molar refractivity of the compound and/or the ability of the compound to bind zinc and/or inhibit a metalloprotease involved in the remodelling event.
  • a method of selecting a chelating agent as a candaidate inhibitor of invertebrate remodelling events from a collection of metal chelating agents comprising selecting metal chelating agents that have at least two polar atoms capable of simultaneously coordinating with a metal ion and
  • the clogP of a metal chelating agent may be calculated from its logP value using a clogP program, for example, the program provided by Biobythe.
  • LogP values may be obtained from the literature or may be calculated from a measured partition co-efficient between n-octanol and water.
  • the molar refractivity of a metal chelating agent may be calculated using the CMR (calculated molar refractivity) software program from Biobythe.
  • the metal chelating agent is selected to inhibit a zinc-metalloprotease enzyme involved in a invertebrate remodelling event.
  • the chelating agent is further accessed for its ability to bind zinc ions.
  • the ability of a metal chelating agent to bind zinc ions may be determined by determining the association constant (logKb) of the metal chelating agents for zinc.
  • the association constant may be determined from the literature using methods know to those skilled in the art.
  • metalloprotease enzyme is a zinc-metalloprotease enzyme
  • metal chelating agents having an association constant for zinc of greater than 5.0 are selected.
  • the metalloprotease to be inhibited includes a metal ion other than zinc, for example, Mg ++ , Cu ++ or Fe ++ .
  • the association constant of the metal chelating agent for that metal ion may be assessed and metal chelating agents having the greatest association constants selected.
  • Identification of suitable metal chelating agents may further comprise testing the compound in a biological assay.
  • a suitable biological assay preferably comprises exposing a control sample of pests in which the remodelling event may occur to a control buffer solution or control formulation whilst at the same time exposing a test sample of pests in which the remodelling even may occur to a solution or formulation comprising a test compound.
  • a compound that is effective in inhibiting a remodelling event in a pest is identified when the remodelling event is observed in the pests of the control sample or formulation and the remodelling event is not observed in the test sample of pests.
  • the remodelling event may occur in a pest selected from the group consisting of louse, flea, tick, fly, mite and other biting or blood-sucking pests and further includes pests that live inside a mammalian host such as trematodes, nematodes and cestodes.
  • the remodelling event may occur in pests which infest plants such as caterpillars, moths, butterflies and soil nematodes.
  • the remodelling event occurs in a pest that infests an environment, such as a termite egg or house dust mite egg.
  • the control buffer solution may include, but is not limited to, sterile phosphate buffered saline or water or an organic solvent.
  • the compound tested is preferably a metal chelating agent.
  • egg hatching is observed when the hatchflap or operculum of the egg opens and shortly thereafter the larvae or nymph begins to emerge.
  • the head appears first followed by the thorax to which the legs are attached. Finally, the abdomen comes out and the nymph moves free from the egg.
  • the eggshell then remains cemented to the hair shaft.
  • a metal chelating agent test compound may be identified as suitable for use in the invention if the eggs exposed to the control buffer display a high level, for example 70-100%, of hatching whereas the egg exposed to a test metal chelating agent display a low level, for example 0-30%, egg hatching, especially where 100% inhibition of egg hatching occurs.
  • the pLD 50 of the selected metal chelating agent is greater than 2, preferably greater than 3, and more especially greater than 4.
  • an invertebrate at a particular life stage for example, a cyst, a larvae, a cocoon, a pupa, a nymph or an adult may be exposed to a test metal chelating agent in a carrier and the occurrence of a remodelling event such as excystment, exsheathment, apolysis, ecdysis or metamorphosis observed and compared to a control group of invertebrates exposed to carrier in the absence of test metal chelating agent.
  • a remodelling event such as excystment, exsheathment, apolysis, ecdysis or metamorphosis
  • At least one metal chelating agent in the manufacture of a composition for inhibiting a protease enzyme involved in invertebrate remodelling events or for treating or preventing pest infestation, wherein the at least one chelating agent has at least two polar atoms capable of simultaneously coordinating with a metal ion and
  • the pest is one infesting a plant host. In another embodiment, the pest is one infesting a domesticated animal. In yet another embodiment, the pest is one infesting a human. In a further embodiment, the pest is one infesting an environment.
  • agents comprising at least one metal chelating agent as described herein, for inhibiting a protease enzyme involved in invertebrate remodelling events or for treating or preventing pest infestation.
  • the compounds tested for efficacy as pesticides may be part of a set or library of compounds, which may be a diverse set or library or a focused set or library, as will be clear to the skilled person.
  • the libraries that may be used for such screening can be prepared using combinatorial chemical processes known in the art or conventional means for chemical synthesis. Collections of compounds of the formula (I) and/or formula (II) which can be synthesized manually or in a semiautomated or fully automated manner. In this case, it is possible, for example, to automate the procedure for the production of such compounds, work-up or purification of the products or of the intermediates generally as described in, for example, by S. H.
  • the compounds used in the invention are not bestatin.
  • the compound used in the invention is not 1,10-phenanthroline. In other embodiments, the compound used in the invention is not 2,2′-bipyridine.
  • the mechanism of lice egg hatching was assessed under a dissecting microscope.
  • Female clothing lice were fed for half an hour on a rabbit before being transferred to a petri dish containing human hair.
  • the petri dish was then placed in an incubator at 32° C.; 32% relative humidity.
  • Within 5 hours of feeding the female lice begin to lay their eggs.
  • Each female lays up to 10 eggs at a sitting.
  • the eggs develop over the next 7-9 days.
  • Within the last 12 hrs prior to hatching the following changes were observed.
  • the eyes of the developing embryo could be clearly detected inside the egg with the developing embryo orientated so that it has its head is adjacent to the hatch flap or operculum. The embryo can be observed moving within the egg. Hatching takes place when the operculum opens and shortly thereafter the embryo begins to emerge.
  • sample 1 A number of other samples were also collected as described. Sample 2 was collected by removing the unhatched louse eggs from four hairs that were approximately 3 cm long, cutting the hair into 0.5 cm lengths, and placing them into a microfuge tube containing 20 ⁇ l of distilled water and incubating at 32° C. for 30 minutes.
  • Sample 3 was collected as for sample 2, but the hair was placed in a tube containing 10 mL of 1% sodium hypochlorite for 1 minute followed by five 1 minute washes in 25 mL of distilled water to remove the sodium hypochlorite before being incubated in a microfuge tube containing 20 ⁇ l of distilled water and incubated as for Sample 2.
  • Sample 4 was collected from unhatched eggs which were removed from the hair and washed with 1% sodium hypochlorite and incubated in 20 ⁇ l of distilled water in the same manner as the hair in Sample 3.
  • Sample 5 was collected from eggs that were within 24 hrs of hatching which were washed with 1% sodium hypochlorite, then returned to the incubator at 32° C.
  • Protease activity in the ESWs from louse eggs was also examined using two-dimensional gel electrophoresis. It was necessary to collect large numbers of freshly hatched egg shells. Following egglaying onto pieces of cloth, adult female lice were removed and the cloth with the eggs washed with 1% sodium hypochlorite as for collection of ESWs. The eggs were then returned to the incubator and permitted to hatch. Typically 100 to 500 hatched egg shells were collected (0-2 hours post hatching), placed in a microfuge tube containing 200 ⁇ l of distilled water, incubated and sample treated as described above.
  • ESWs were resuspended in rehydration buffer (8 M Urea, 2% 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate 2% Immobilised pH gradient buffer 3-10 (Amersham Phamacia Biotech, Uppsala, Sweden)) and used to rehydrate 7 cm Immobiline Dry strips pH 3-10 (Amersham Bioscience) overnight.
  • Strips were transferred to the Multiphor II (Pharmacia, Sweden) apparatus, electrophoresed in the first dimension at 200 V for 1 min, increasing to 3,500 V over the next 90 min followed by 65 min at 3,500 V, equilibrated (6 M urea, 30% glycerol, 50 mM Tris pH 8.8 and 2% SDS) and then run on a 10% SDS-PAGE gel containing 0.1% gelatin for the second dimension. The gel was then run and developed as previously described.
  • protease activity was detected in washings from unhatched eggs within 12 hours of hatch (Sample 1) in the higher molecular weight region of the gel, above 50 kDa ( FIG. 1A , Lane 1).
  • a similar pattern of protease activity was detected in the washings taken from human hair samples following the removal of the louse eggs (Sample 2) ( FIG. 1A , Lane 2).
  • treatment of the hair with 1% sodium hypochlorite prior to collecting the washings (Sample 3) completely removed the protease activity ( FIG. 1A , Lane 3).
  • Hypochlorite treatment was also able to remove the extraneous proteases from unhatched louse eggs (Sample 4) ( FIG. 1A , Lane 4).
  • Hypochlorite was used to treat unhatched eggs prior to the collection of ESWs for all subsequent protease analyses.
  • Two-dimensional gelatin SDS-PAGE was used to more accurately assess the number of protease species present in the louse ESWs.
  • Each of the three main regions of protease activity in the one-dimensional gelatin SDS-PAGE ( FIG. 1B ) resolved to a number of distinct proteases when analysed by two-dimensional gelatin SDS-PAGE ( FIG. 3A ).
  • the proteases present in the louse ESWs with activity in the 25-30 kDa molecular weight range resolved to at least seven distinct proteases with isoelectric points in the neutral to alkaline pH range, whereas the band of protease activity around 50 kDa resolved to at least eleven distinct protease regions with isoelectric points in the acidic to neutral pH region.
  • the regular banding pattern of the proteases in the 50 kDa region suggests that they may be related in some manner. At least five proteases with molecular weights above 75 kDa were also observed.
  • the inhibitors used were the serine protease inhibitor PMSF (5 mM), the metalloprotease inhibitors 1,10-phenanthroline (10 mM) and EDTA (ethylenediamine tetraacetic acid) 10 mM, the aspartic protease Pepstatin (5 ⁇ M) and the cysteine inhibitor E-64 (10 ⁇ M).
  • the gel strips were then incubated in 0.1M Tris/HCl containing 1 mM CaCl 2 pH 8 containing the different protease inhibitors for 3 hrs at 37° C., before being stained in Coomassie blue and destained as previously described.
  • a similar approach may be used to characterise proteases in egg shell washings of other thin membrane eggs from different pests.
  • a similar approach may be used to develop in vitro bioassays for measuring other remodelling events that include, but are not limited to, thin membrane egg hatching.
  • Lice eggs were laid onto cloth as described above. Five days post laying the cloth containing lice eggs was removed and immersed in a 1% sodium hypochlorite solution before being washed extensively in distilled water and blotted dry on tissue paper. Lice eggs were counted under a dissecting microscope and the cloth cut into batches of between 10-30 eggs with 3-5 replicates used per treatment. The cloth containing lice eggs was then immersed in a protease inhibitor solution for a period of 10 minutes, placed on tissue paper for 1 minute to dry before being transferred to a clean petri dish and incubated until hatching. The eggs were observed at regular time intervals for evidence of eggs hatching over the next 1-2 days by which time the control eggs had hatched.
  • Protease inhibitor solutions were typically prepared as stock solutions and added fresh at the appropriate concentration. Specifically a stock solution was prepared as follows: 1,10-phenanthroline (200 mM in methanol). In addition, the equivalent levels of the solvent were added to the non-inhibitor containing controls eggs to test for any buffer alone effects. Percentage hatch inhibition was calculated as the percentage reduction in egg hatch compared to the untreated control. The untreated control was assigned a percentage hatch of 100%.
  • 1,10-phenanthroline a metal chelating agent and a metalloprotease inhibitor significantly inhibited egg hatching in lice at 10 mM while at 1 mM the level of inhibition was approximately 30% compared to that of the controls (refer to FIG. 4 ).
  • a number of metal chelating agents with potential for inhibiting the remodelling events associated with thin membrane egg hatching in lice were analysed by comparing their percentage ovicidal activity in lice at different concentrations, their logit calculated pLD 50 , Pref pLD 50 , Activity class, clogP and molar refractivity were determined. The results are shown in Table 2.
  • a control (ie no inhibitor treatment) group of eggs was also included.
  • the eggs were removed from the inhibitor, blotted dry on tissue paper, placed at 32° C., 70% RH and left to hatch.
  • a second group of eggs, (48 hours old) were treated as previously described and also left to hatch. This process was repeated at 24 hour intervals on head lice eggs up to 120 hours post laying. This method of assaying inhibitors more closely mirrors the field situation where lice eggs will be at various stages of development on the head and permits the inhibitory effects to be observed on these different stages of the parasite.
  • NIX® Pfizer active ingredients, 1% permethrin.
  • Results from the testing of 3 commercial pediculicides indicate that they displayed inconsistent levels of ovicidal activity across the different stages of lice egg hatching. Whereas the compound 1,10-phenanthroline was highly effective at inhibiting lice egg hatching.
  • the ovicidal properties of two major commercial head lice products were evaluated in the standard lice egg-hatching assay.
  • the 2 commercial head lice products were as follows:
  • Table 9 indicates that the metal chelator 6,6′ dimethyl-2,2′ dipyridyl was able to inhibit egg hatching in Plutella xylostella in a dose dependent manner, with strong ovicidal effects evident at both 10 and 1 mM.
  • the metalloprotease inhibitor/metal chelator, 1,10-phenanthroline was also able to significantly inhibit egg hatching of this insect at 10 mM.
  • Tables 10 and 11 show the effects of exposing Plutella xylostella eggs to selected dipyridyl compounds on egg hatching relative to controls.
  • the results show a dose dependent effect for 6,6′-dimethyl-2,2′ dipyridyl with both 10 and 1 mM being effective at inhibiting egg hatching of the Plutella eggs. At 0.1 and 0.01 mM, there was no observable effects on egg hatching.
  • Example 12 confirm the results shown in Example 12 for this compound.
  • both 5,5′-dimethyl-2,2′ dipyridyl and 4,4′-dimethyl-2,2′ dipyridyl were able to significantly inhibit egg hatching at both 10 and 1 mM.
  • Lannate® (Crop Care Australasia Pty Ltd) containing methomyl as an active compound was chosen as a comparative control.
  • the ovicidal activity of 2-acetyl-1-tetralone against H. armigera eggs was assessed as described in Example 14.
  • the compound was dissolved in 100% diethoxyglycol and diluted to a final concentration of 1% in water.
  • the results are given in FIG. 6 and show that 2-acetyl-1-tetralone displayed strong ovicidal efficacy at 2 mM with efficacy declining at 1 mM.
  • Table 14 indicates that the metal chelating compound 2-(2-pyridinyl)quinoline was able to inhibit egg hatching in Plutella xylostella at 10 mM.
  • Results presented in Table 15 indicate that the addition of the divalent metal ions in the form of Fe in FeSO 4 was able to reverse the effects of the metal chelating agent 6,6′-dimethyl-2,2′-dipyridyl.
  • the results indicate that the reversal of the inhibitory effects of 6,6′-dimethyl-2,2′-dipyridyl are due to Fe replacing the action of this inhibitor as opposed to a simple dilution of the inhibitor by the FeSO 4 . This effect is indicated by the finding that exposure of the eggs to MeOH alone post exposure to the inhibitor still resulted in a significant degree of inhibition of egg hatching.
  • Results presented in Table 16 indicate that the addition of the divalent metal ions in the for of Fe in FeSO 4 was able to reverse the effects of the metal chelating agent 5,5′-dimethyl-2,2′-dipyridyl.
  • the results indicate that the reversal of the inhibitory effects of 5,5′-dimethyl-2,2′-dipyridyl are due to Fe removing the action of this inhibitor as opposed to a simple dilution of the inhibitor by the FeSO 4 . This effect is indicated by the finding that exposure of the eggs to MeOH alone post exposure to the inhibitor still resulted in a significant degree of inhibition of egg hatching.
  • the ovicidal efficacy of 2-acetyl-1-tetralone was also tested against Plutella eggs.
  • the compound was dissolved in diethoxyglycol and then diluted to a final concentration of 1% diethoxyglycol containing 1 mM 2-acetyl-1-tetralone and tested in the same manner as in Example 13.
  • the results of this assay are given in FIG. 7 and indicate strong ovicidal efficacy of this compound against Plutella at 2 and 1 mM with no inhibition observed at 0.5 mM.
  • the ovicidal efficacy of 2,2′,6,2′′-terpyridine and 5,5′-diethyl-2,2′ dipyridyl was also tested against Plutella eggs.
  • the compounds were dissolved in diethoxyglycol and then diluted to a final concentration of 1% diethoxyglycol containing 1 mM 2,2′,6,2′′-terpyridine or 1 mM and 0.1 mM 5,5′-diethyl-2,2′-dipyridyl and tested in the same manner as in Example 13.
  • the results of this assay are given in Table 17 and indicate complete inhibition at 1 mM for both compounds. Partial inhibition of egg hatching was observed at 0.1 mM 5,5′-diethyl-2,2′-dipyridyl.
  • ovis eggs were collected from the wool of sheep that were infested with this parasite.
  • the eggs were collected using forceps and with the aid of a dissecting microscope and placed in 24 well tissue culture plates in duplicate lots of 10 eggs per replicate.
  • the eggs were then exposed to either methanol alone (control) or the test compounds for either 10 minutes or 1 minute before being removed from the wells and placed into individual glass vials containing a diet at the base of the tube.
  • the tubes were placed in plastic containers containing a salt solution (to keep humidity constant at 68%) and the containers maintained at a temperature 32° C.
  • the eggs were monitored for hatching over the following 12 days and % hatch inhibition determined in comparison to the controls.
  • the gastrointestinal parasite Haemonchus contortus is a major pathogen of sheep throughout the world. The parasite survives through the ability of the adult worms to attach to the abomasal mucosa of the sheep and draw blood.
  • One adult female can take in approximately 0.1 ml blood per day.
  • the adults live can live for many months with the females producing several hundred eggs per day and infected animals shedding upwards of several thousand eggs per gram of faeces per day onto pasture.
  • the eggs hatch after 1-2 days depending on weather conditions and following two moults infective L3 larvae appear on the pasture and are consumed by the host.
  • FIG. 8 indicates that 5,5′-dimethyl-2,2′-dipyridyl was potently ovicidal at 180 and 18 ug/mL (equivalent to 1 and 0.1 mM of the active respectively).
  • a comparison of the ovicidal efficacy of 5,5′-dimethyl-2,2′-dipyridyl to the commercial product ivermectin indicated that 5,5′-dimethyl-2,2′-dipyridyl was in the order of 10 ⁇ more effective at inhibiting H. contortus egg hatching compared to ivermectin ( FIG. 8 and FIG. 9 ).
  • a filter paper (90 mm diameter) was taken and placed in a Petri dish of the same dimensions. The filter paper was then wetted throughout with the test compound, using a small air pump sprayer. The wetted filter paper was then allowed to dry in free flowing air. 200 mg dust mite medium, containing roughly 500 mites/g, was placed on the filter paper and the number of mites counted under a microscope. The arena was then left for 2 weeks on an incubator at 25° C. and 75% RH. After 2 weeks the number of mites in the arena was counted for a second time. This experiment was repeated 3 times with the test compound and a further 3 times using water as a control. The results are presented in table 19.
  • the mite populations on the treated filter papers showed a small decline over the 2 week period. This may be due to prevention of eggs from hatching and/or to effects on oviposition of the female mites. In contrast, the control treatments showed small increases in mite populations, suggesting a lower mortality of adult mites and no adverse effect on egg viability.
  • Cat flea eggs were exposed for 10 minutes to 10 mM 5,5′-dimethyl-2,2′-dipyridyl and then removed from the solution and placed in an incubator and left to hatch. A group of house dust mite eggs were exposed to the vehicle only and were used as controls. A third group remained untreated. Subsequently the eggs were examined and the percentage of eggs that successfully hatched compared to the controls determined.
  • Bed bug eggs were exposed for 10 minutes to 10 mM 5,5′-dimethyl-2,2′-dipyridyl and then removed from the solution and placed in an incubator and left to hatch. A group of house dust mite eggs were exposed to the vehicle only and were used as controls. A third group remained untreated. Subsequently the eggs were examined and the percentage of eggs that successfully hatched compared to the controls determined.
  • Third stage H. contortus larvae were exposed to varying concentrations of 5,5′-dimethyl-2,2′-dipyridyl and the effects on moulting from L3 to L4 examined.
  • the larvae were either exsheathed (their L2 sheath was removed chemically) or unexsheathed (their L2 sheaths were intact).
  • the larvae were exposed to varying concentrations of 5,5′-dimethyl-2,2′-dipyridyl added to their culture media of DMEM and the effects on larval survival monitored over time. Following a 30 minute incubation at 37° C., greater than 90% of the exsheathed larvae exposed to 1 and 0.5 mM of the compound appeared dead.
  • 5,5′-dimethyl-2,2′-dipyridyl was formulated and evaluated in the standard body louse egg assay.
  • Body louse eggs (15-30 per replicate) of varying ages were exposed for 10 minute to the test solutions or a placebo or left untreated, followed by a 1 minute water wash and blotted dry. The eggs were then incubated at 30° C. over the following 10 days post treatment and the percentage of eggs that successfully hatched was determined (Table 20). The results show a strong dose dependency of ovicidal activity when the compound 5,5′-dimethyl-2,2′-dipyridyl is formulated and applied to body louse eggs.
  • HT compound 1 (5,5′diethyl-2,2′-dipyridyl 6 0 0 0 0 0 10 mM (200 L/Ha) No Wetting Agent 7 0 0 0 0 0 0 8 0 0 0 0 0 0 9 0 0 0 1 1 1 10 0 1 1 0 1 2 Treatment 3.
  • HT compound 2 (5,5′diethyl-2,2′-dipyridyl) 11 0 0 0 0 0 10 mM (200 L/Ha)* 12 0 0 0 0 0 0 13 0 1 1 2 2 2 14 0 0 3 3 3 3 3 15 0 0 1 1 1 1 Treatment 4.
  • HT compound 2 (5,5′dimethyl-2,2′-dipyridyl) 16 0 0 0 0 1 10 mM (200 L/Ha)* 17 0 0 0 0 0 0 18 0 0 0 0 0 19 0 0 0 1 1 1 20 0 0 1 2 2 2 Treatment 5.
  • HT compound 2 (5,5′dimethyl-2,2′-dipyridyl) 21 0 0 0 1 1 1 20 mM (200 L/Ha)* 22 0 0 0 0 0 0 23 0 0 0 0 0 0 24 0 0 0 0 0 0 25 0 0 0 0 0 0 Treatment 6.
  • Negative control 26 2 5 8 8 8 10 (Vehicle only, Placebo)* 27 0 3 8 10 10 10 28 0 1 9 9 9 10 29 0 0 2 7 8 10 30 0 5 9 10 10 10 Treatment 7.
  • Negative control 31 3 0 9 10 10 10 (Water) 32 3 0 10 10 10 10 33 0 5 8 10 10 10 34 0 5 6 10 10 10 35 0 0 10 10 10 10 *All these formulations contained wetting agent at 0.3 ml/L

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US9839631B2 (en) 2003-07-16 2017-12-12 Dr. Reddy's Laboratories, S.A. Methods and compositions for controlling ectoparasites
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